Seroprevalence of Toxoplasma gondii and Associated Risk Factors among Pregnant Women in Algeria

ABSTRACT. Toxoplasma gondii is an obligate intracellular protozoan parasite affecting all warm-blooded vertebrates, including humans. Infections in humans can lead to severe clinical manifestations in pregnant women and immunocompromised patients. The aim of the present study was to assess seroprevalence of T. gondii infection and to identify the associated risk factors among pregnant women from southern Algeria. A cross-sectional study was conducted from March 2021 to May 2022 among 1,345 pregnant women. A structured questionnaire was used to obtain information on risk factors associated with infection. Participants were screened for anti T. gondii IgG and IgM antibodies using the enzyme-linked fluorescent assay. The overall IgG and IgM seroprevalence was 13.6% and 0.89%, respectively. A significant association was found between seroprevalence of anti T. gondii IgG and history of spontaneous abortion (P = 0.016). Moreover, an increasing trend of seroprevalence was detected in the autumnal season (P = 0.030), and eating raw or undercooked meat was found to be significantly associated with anti–T. gondii IgM seropositivity (P = 0.002). Multivariate analysis showed that women who had experienced spontaneous abortion, regularly ate undercooked meat, and used bottled water in summer were more likely to contract infection with T. gondii. The majority (86.4%) of the studied pregnant women were serologically negative for toxoplasmosis and hence were susceptible to primary acute infection during pregnancy and possible fetal anomalies. Therefore, health education and awareness of the disease and its transmission to women, especially during pregnancy, is imperative.


INTRODUCTION
Toxoplasmosis is a common worldwide parasitic zoonoses, caused by an obligate intracellular protozoan parasite, Toxoplasma gondii. 1 Although most infections are asymptomatic, infection can have severe consequences if pregnant women become infected.Toxoplasma gondii is capable of crossing the transplacental barrier from a primo-infected pregnant woman to her fetus, 2 leading to congenital toxoplasmosis, although the risk of fetal transmission depends on the exact timing of exposure during pregnancy.Consequently, depending on the trimester of pregnancy, infection with T. gondii can cause not only miscarriage or stillbirth but also serious and progressive visual, hearing, motor, and cognitive complications for the child. 3Therefore, early diagnosis of toxoplasmosis crucial to enable treatment in sufficient time to minimize transplacental transmission.In the majority of cases, toxoplasmosis is an asymptomatic disease in adults, but it can have devastating consequences in immunocompromised patients and nonimmunized pregnant women. 4,5Toxoplasma gondii is commonly transmitted to humans by accidental ingestion of the oocyst stages of the parasite in water or food, in soil contaminated with feces from infected cats, or by eating raw or undercooked meat containing tachyzoites or bradyzoites. 6,7nfection is usually asymptomatic, thus it is most often detected by serological testing in pregnant women, although clinical analysis can contribute in subjects showing acute signs of infection.Toxoplasma-specific antibodies IgM and IgG can be detected in the serum of pregnant women within 1 to 2 weeks of exposure to infection. 8In patients with Toxoplasma infection, it is important to establish whether they have an acute or chronic infection.Additional screening through the avidity test of T. gondii-specific IgG antibodies 9 and polymerase chain reaction analysis of the amniotic fluid are usually used for greater certainty. 10A positive diagnosis of acute infection in a pregnant woman indicates a high risk of potential transmission of the parasite to the fetus, and this should trigger treatment to prevent vertical transmission. 11revalence of toxoplasmosis is known to vary widely between countries. 12In Algeria, seroprevalence of toxoplasmosis during pregnancy has been poorly reported. 10,13,14he overall seroprevalence of T. gondii infection ranges from 25% to 52.6%, with seroprevalence of recent Toxoplasmaspecific IgM antibodies ranging from 1.1% to 1.6% 13,14 and prevalence of acute infection for both IgG and IgM antibodies ranging from 4.8% to 43.8%. 10,14However, there is scarcity of information on the risk factors for contracting T. gondii infection among pregnant women in Algeria, and there are no published reports on T. gondii infection in Laghouat province.This study therefore sought to determine seroprevalence of T. gondii and to identify the potential risk factors associated with T. gondii infection among pregnant women in Laghouat province, southern Algeria.

MATERIALS AND METHODS
Study area.This study was carried out in the province of Laghouat, situated in the central south of Algeria, 400 km to the south of the capital Algiers, between latitude 33 48 9 north and longitude 02 53 9 east.It is characterized by agropastoral activity and covers 25,052 km 2 , with an estimated population of 716,219 inhabitants in 2020.In total, 12,820 pregnant women attend the maternity clinic per year with a birth rate estimated at 98.8%, and a predicted 10-year population growth rate of 3.8% (unpublished observations, monograph of the wilaya of Laghouat).Daily temperatures average 25 C in winter months and .40C in summer, and the average annual rainfall is 151.21 mm (Unpublished data, Bouchetata, 2018).Agricultural activities such as livestock husbandry and breeding (sheep, goats, cattle, horses, camels, and chicken) are the main economic activities in the rural population, and water is available through boreholes and taps supplied by wells or public water delivery systems (Unpublished data, Directorate of Agricultural Services, 2016).
Selection of subjects and study design.A crosssectional survey was conducted among all apparently healthy pregnant women who agreed to participate in the study from March 2021 to May 2022 and who had been referred to three private laboratories conducting medical analyses in Laghouat province.In Algeria, serological screening of pregnant women for T. gondii infection is routinely requested by gynecologists.Data were collected by laboratory personnel using a structured questionnaire constructed in French and translated into Arabic, the local language.Pregnant women (N 5 1,345) were interviewed on the day of blood sample collection to acquire data about the potential risk factors for toxoplasmosis including age, symptoms, residential area, stage of pregnancy, any previous experience of abortion, contact with cats, water consumption, consumption of undercooked meat, and exposure to soil.Questionnaires in Arabic guided the face-to-face interviews conducted by the investigators, who briefed the participants about the study objectives.Questions were answered orally by the interviewees, and the answers were recorded by the investigators for subsequent conversion into digital format and addition to the database used for statistical analysis.Incomplete patient records and, to avoid bias in the results, cases of women who returned for followup to earlier examination were excluded from this study because the data for analysis were restricted to cases of pregnant women visiting the laboratory for the first time.
Sample collection and ethical approval.Ethical clearance for the study was obtained from the Faculty of Science of Nature and Life, Djelfa University, Algeria (Ref:AT04/ E.V.E.S.).The study objectives and procedures for sample collection were explained to participants, and informed written consent was obtained.Approximately 5 mL of venous blood were collected from each pregnant woman in a tube coated in EDTA.Each tube was centrifuged at 3,000 3 g for 5 minutes, and the collected plasma was tested directly.
Serological testing.Serological diagnosis of toxoplasmosis was performed assessing presence of both Toxoplasmaspecific IgG and IgM antibodies in participants' sera by the three private medical laboratories in Laghouat province, all using identical techniques.The sera were analyzed using a standard enzyme-linked fluorescent assay using the Vitek Immuno-Diagnostic Assay System (VIDAS Toxo IgG II and IgM, bio M erieux-Marcy-l'Etoile, France) run in a VIDAS fully automated immunoanalyzer.The system was calibrated and standard operating procedures were implemented following the manufacturer's instructions.A titer of IgG anti-Toxoplasma antibody $8 IU/mL was considered positive in this study, equivocal results were defined as IgG values ranging from 4 to 8 IU/mL, and negative results were defined as IgG values of ,4 IU/mL.Samples were considered reactive for IgM when the antibody concentration was greater than or equal to an index of 0.65, nonreactive when the concentration was ,0.55 index, and undetermined when the concentration was between 0.55 and 0.65 index.

STATISTICAL ANALYSES
For data subsets, prevalence values (percentage of subjects with positive IgG or IgM titers) are given with 95% confidence limits (CL 95 ) in the text and 95% CIs in figures, and calculated in bespoke software based on the tables of Rohlf and Sokal. 15Odds ratios 1 95% CLs were calculated for levels within each factor, using one level as the reference point in each case.
Prevalences were analyzed using maximum likelihood techniques based on log linear analysis of contingency tables in the software package IBM SPSS, v. 28.0 (IBM Corp., Armonk, NY).This approach is based on categorical values of factors of interest, which are used not only to fit hierarchical log-linear models to multidimensional crosstabulations using an iterative proportional-fitting algorithm but also to detect associations between factors, one of which is the presence or absence of IgG or IgM.We first fitted models with presence/absence of IgG positivity and each of the explanatory factors in turn ), year (two levels, 2021 and 2022), trimester of pregnancy (three levels, first, second, and third trimester), history of abortion (two levels, experienced spontaneous abortion or not), season (four levels, spring, summer, autumn, and winter), location (two levels, rural or urban), use of water (bottled or tap), possession of a pet cat (two levels, yes or no), consumption of meat (well-cooked or poorly cooked), and possession of a garden (two levels, yes or no).
Next, we fitted multifactorial models in three stages as described previously 16 because of the number of factors involved.First, we fitting a model with intrinsic explanatory factors, then one with extrinsic factors, and finally we fitted a model with all the factors that had shown P-values of ,0.3 in the first two stages.By employing the backward selection procedure in SPSS, we simplified each model until only significant terms remained.For each level of analysis in turn, beginning with the most complex model, involving all possible main effects and interactions, those combinations that did not contribute significantly to explaining variation in the data were eliminated in a stepwise fashion beginning with the highest level interaction (backward selection procedure).A minimum sufficient model was then obtained, for which the likelihood ratio of x 2 was not significant, indicating that the model was sufficient in explaining the data.The importance of each term in interactions involving presence or absence of IgG or IgM in the final model was assessed by the probability that its exclusion would alter the model significantly and these values are given in the text, assessed by a likelihood ratio test between models with and without each term of interest.

Sociodemographic characteristics.
A total of 1,345 pregnant women aged between 19 and 44 years, with a mean age of 29.37 were enrolled during the study period.Approximately one-quarter of the study population were in the age range of 28-31 years (24.6%).One hundred twentynine (10%) of the participants had a history of spontaneous abortion.Of all participants, 753 (55.9%) were urban and 592 (44.1%) were rural residents.With regard to the gestational age, it was found that 66.5% of the women were in the first trimester of pregnancy, 27.3% were in the second trimester and 6.2% were in the third trimester.
Seroprevalence of T. gondii infection.The prevalence of specific anti T. gondii IgG was 13.6% (95% CI: 11.77-15.44),and that of IgM considerably lower at 0.89% (95% CI: 0.461-1.558).Prevalence by levels within the factors, together with odds ratios and comparison of odds ratios to the first listed level within each factor, are summarized in Table 1 (IgG) and Table 2

(IgM).
Risk factors associated with IgG anti T. gondii infection.The prevalence of IgG positive samples was remarkably consistent across eight age classes (x 2 7 5 3.73, P 5 0.81) and the trimesters of pregnancy (x 2 2 5 1.31, P 5 0.52).As for the obstetric variables, the pregnant women who had a spontaneous abortion history showed a significantly higher seroprevalence of T. gondii infection (20.9% versus 12.8%; x 2 1 5 5.820, P 5 0.016).Although there was no overall significant effect of season of the year (x 2 3 5 3.92, P 5 0.27), based on comparison of odds ratios, prevalence was significantly, but not substantially, higher in autumn compared with spring (Table 1).There was no significant difference in prevalence between the 2 years of the study (x 2 1 5 0.034, P 5 0.85), whether subjects lived in rural or urban areas (x 2 1 5 0.32, P 5 0.57), relied on bottled or tap water (x 2 1 5 0.11, P 5 0.74), had pet cats or not (x 2 1 5 0.85, P 5 0.36), consumed well or poorly cooked meat (x 2 1 ,0.001,P 5 0.99), or had access to gardens or not (x and history of abortion) found only one significant main effect (history of abortion, x 2 1 5 5.820, P 5 0.016) but no significant interactions.
Multifactorial analysis in three stages (intrinsic, extrinsic, and behavioral factors, and then factors with P ,0.3 in first two stages) revealed only one complex interaction in a model comprising only the behavioral factors with IgM (water 3 meat 3 garden 3 IgM; x 2 1 5 4.69, P 5 0.030), which was not explored further.However, we next fitted a model with water, meat, and garden and also with age, season, and location because the latter three all showed P-values of approximately 0.2-0.3.This generated three relevant interactions, the most significant of which was meat 3 location 3 IgM (x 2 1 5 12.65, P ,0.001).This is illustrated in Figure 1, which shows that although IgM prevalence was low among urban dwellers irrespective of whether they consumed well or poorly cooked meat, it was higher among rural dwellers who regularly ate poorly cooked meat.The second interaction comprised season 3 water 3 IgM (x 2 3 5 11.41, P 5 0.010).Figure 2 shows that prevalence of IgM positive samples was higher among those using bottled water in the summer months, when prevalence was zero among those using tap water.In other seasons, there was little difference between those using these two types of water resource.The third interaction was garden 3 season 3 IgM (x 2 3 5 7.96, P 5 0.047), but this was only just significant and was not explored further.

DISCUSSION
The overall prevalence of anti-T.gondii IgG antibodies observed in this study was 13.6%, indicating that these women, with positive IgG titers, has experienced infection in the past and probably had acquired resistance to further infection.However, 86.4% of the pregnant women had no IgG antibodies to the parasite and therefore were susceptible to infection if exposed during the course of pregnancy and, depending on the trimester, were capable of transmitting the infection to the fetus.Therefore, serological monitoring is required during each trimester, with prophylaxis to follow throughout the pregnancy in positive cases.
When we compared our findings with those of previous studies reporting prevalence of toxoplasmosis among pregnant women in different cities in Algeria, we found that T. gondii seroprevalence in these other regions, characterized by lower altitudes and with higher humidity, was higher than in the current work (e.g., in Annaba, northeastern Algeria, where prevalence has been reported to be 47.8% 13 and 52.6% 10 ).It is also lower than in Medea, northern Algeria (25%). 14This difference could be attributed to the high relative humidity that typifies northern Algeria (center, eastern, and western), which is likely to enhance oocyst survival in the environment preserving viability for longer compared with in the south of Algeria where the climate is much drier.In addition, our results remain lower than those reported in neighboring North African countries, in Europe, and in Asia: 43% in Morocco, 17 45.6% in Tunisia, 18 50.8% in Libya, 19 40.1% in Egypt, 20 31.3% in France, 21 55.8% in Romania, 22 31% in Turkey, 23 35.1% in Qatar, 24 82.6% in Lebanon, 25 and 21.2% in Yemen. 5However, the results of our survey are similar to those reported in Palestine (17.6%), 26 Sri Lanka (12.3%), 27 and Italy (13.8%). 28Indeed, variation of T. gondii seroprevalence among individuals could be attributed to many factors, including climatic conditions (dry climate, rainfall, temperature, soil type, and altitude).
In our study, a significant association was observed between IgG seroprevalence of T. gondii and history of spontaneous abortion, and similar findings have been reported in studies from Yemen 5 and Egypt. 29Many studies in the literature suggest a relationship between T. gondii infection and spontaneous abortion. 30However, in the present study IgG seroprevalence of T. gondii infection was not influenced by age, trimester of pregnancy, place of residence, water consumption, meat consumption, garden work, or by presence of cats in the household.These results are strongly supported by a study carried out in Denmark in which the authors found that up to two-thirds of infections could not be explained by the presence of any specific risk factor. 31Nevertheless, this finding contrasts with some previous studies, as, for example, in Medea province, where the risk of toxoplasmosis was significantly associated with increased maternal age. 14A similar finding was reported in Qatar. 32In Annaba, Algeria, consumption of undercooked meat and contact with cats were reported as significant risk factors for higher T. gondii seroprevalence. 13Cats have been considered to constitute a high risk factor responsible for transmission of infection to intermediate hosts including humans. 33In Italy, a seroprevalence of 19.1% was reported, and the key factors that were shown to increase the risk of infection were consumption of raw meat, contact with cats, consumption of unwashed vegetables or fruit, and contact with soil. 34n the present study, 12 (0.89%) cases were seropositive for both IgG and IgM antibodies, which suggests acute infections in these individuals.This finding is lower than those reported from previous studies conducted among pregnant women in Algeria, in which seroprevalence of acute infection for both IgG and IgM antibodies ranged from 4.8% to 43.8%. 10,13However, further confirmation by using seroconversion and/or IgG avidity tests was not carried out in the present study.
Generally, IgM antibody is the first immunoglobulin to be synthesized in response to an immunogenic agent and therefore is an indicator of recent infection, which can help in the determination of acute infection.Usually, detectable IgM appears 1 week after exposure to T. gondii, and its levels then rise, peaking after 1 to 3 months.This is then followed by a slow decrease over the next 9 months until T. gondii-specific IgM is no longer detectable.However, one study reported that IgM antibodies persisted and were detectable for more than 2 years in 27% and 9% of women using the immunosorbent agglutination assay and indirect fluorescent antibody test, respectively. 35Therefore, the detection of IgM antibodies is not necessarily a marker of recent infection, 36 and in such cases, positive IgM test results should be confirmed by additional specific serological tests, including the test for IgG avidity 9 and IgA and IgE antibody assessment and/or molecular evaluation. 37Nevertheless, the advantage of avidity testing is that it ensures that unnecessary spiramycin treatment in pregnant women and unjustified long-term follow-up of fetuses and infants are avoided. 38n general, serological screening of pregnant women for T. gondii infection is systematically requested by gynecologists in Algeria, who usually recommend treatment with spiramycin until delivery (at a rate of 1 g orally every 8 hours) as soon as a diagnosis of maternal infection is established or strongly suspected.Spiramycin is given to prevent vertical transmission of the parasite to the fetus and is therefore only indicated before the appearance of a fetal infection.If fetal infection is detected, or strongly suspected, the recommended treatment consists of pyrimethamine 1 sulfonamides 1 folinic acid, replacing spiramycin.
Univariate analysis of risk factors in the current work showed that the consumption of undercooked meat was a significant risk factor associated with seropositivity of T. gondii-specific IgM, which is consistent with studies from Annaba, Algeria, 13 Egypt, 20,39 and Yemen. 5However, unlike the present study, other reports from Nigeria, 40 Saudi Arabia, 41 and Tunisia 42 have failed to identify any significant association between consumption of raw and undercooked meat and T. gondii seropositivity.Indeed, many Algerian women are active and eat lunch outside their homes; therefore, they may contract toxoplasmosis by ingesting contaminated foodstuffs (sandwiches, cold cuts, pât e, and sausage).It is also relevant that several studies have identified the handling of fresh meat before cooking as a significant risk factor with a high likelihood of accidental contamination by meatborne stages of T. gondii. 5,43Therefore, a health education program to increase women's knowledge about toxoplasmosis, especially of the sources of infection and modes of transmission, is highly recommended.Preventing infection, notably by avoiding eating raw or undercooked meat, and proper handwashing, particularly after handling or processing raw meat during pregnancy, are all to be encouraged.
Multifactorial analysis of data in the current study enabled identification of more complex interactions between risk factors and T. gondii seroprevalence.Our analysis revealed that seropositivity of T. gondii IgM among pregnant women who consumed undercooked meat was higher if they came from a rural environment rather than from urban locations.The rural population of Laghouat province is mainly engaged in agricultural activities such as livestock husbandry and breeding (sheep, goats, cattle, horses, camels, and chicken), so the high density of domestic animals in rural areas, as well as the favorable environmental conditions for T. gondii oocysts to sporulate, both contribute to the risk of infection.It is also relevant that people from this region are known for their dietary preference of meat consumption and, perhaps not surprisingly given these behavioral characteristics of rural communities in the region, the consumption of undercooked meat leads to a higher prevalence of toxoplasmosis.In a seroepidemiological survey of toxoplasmosis conducted among domestic animals elsewhere in Algeria, the highest prevalence of toxoplasmosis was reported in goats (33.61%), followed by sheep (22.57%) and then cattle (20.04%). 44hese findings suggest that eating or handling raw infected sheep and goat meat, drinking their raw milk, or tasting any of these during cooking may increase the risk of Toxoplasma infection in pregnant women.Also, the preferred habit of eating some traditional Algerian milk products, locally known as Jben cheese, which is made from raw ovine, bovine, and caprine milks and traditional meat meals such as shawarma and brain tissue, may also contribute to and exacerbate the risk of contracting toxoplasmosis in the study population.Further studies to investigate the occurrence of toxoplasmosis in animals in the province are urgently required.
Significantly, increased T. gondii seroprevalence was also found among those using bottled water compared with those using tap water in the summer months, and both sources of water in other seasons.Waterborne transmission of T. gondii has been reported in different parts of the world, including Yemen, 44 Nigeria, 45 Brazil, 46 and Ethiopia. 47Our results may be explicable by the climatic conditions in Laghouat province, which are cooler and drier in winter but warmer and relatively humid in spring and autumn and extremely hot in the summer.Perhaps, stored bottled water provides a more conducive environment for the oocysts of T. gondii, enabling them to remain infectious in water for longer periods over the summer months.
We do acknowledge some limitations in our study.First, a cross-sectional study design does not allow inference of causal relationships between Toxoplasma infection and the significant risk factors.Second, this study was based on a limited population of pregnant women attending only three private laboratories in Laghouat province, which may not represent the general population in the country.Third, a follow-up serological study of all IgG 2 /IgM 1 participants was not conducted to confirm seroconversion to IgG and IgG avidity tests were not conducted in these groups of participants.
In the current study, seroprevalence of toxoplasmosis in pregnant women in Algeria was low compared with that in many other studies.Given the presence of T. gondii in the local environment and 86.4% seronegativity among pregnant women, the risk of primary acute infection during pregnancy was not high but is nevertheless deserving of attention because of the potentially adverse effects on the fetus.Rural dwellers who frequently ate undercooked meat, women with a history of spontaneous abortion, and consumption of tap water in spring and autumn months or bottled water in the summer were associated with a significantly higher risk of contracting T. gondii infection.Antenatal screening of pregnant women and health educational programs about the transmission routes and risk factors for contracting toxoplasmosis in rural areas are strongly recommended.

FIGURE 1 .FIGURE 2 .
FIGURE 1. Prevalence of IgM positive samples in urban and rural areas, among subjects who consumed well-cooked or poorly cooked meat.

TABLE 1
21 5 1.41, P 5 0.24).Multifactorial analysis of intrinsic factors (age class, stage of trimester and history of abortion plus presence/absence of IgG positivity) revealed that only history of abortion was significant.Multifactorial analysis of extrinsic and behavioral factors did not reveal any significant interactions, and a final model in which we included only those factors for which P-values in the first two stages were #0.3 (garden, season, Percentage of subjects showing positive anti Toxoplasma gondii IgG values in data subsets corresponding to the risk factors that were implemented in the study and odds ratios comparing each level to the first listed within each respective factor

TABLE 2
Percentage of subjects showing positive anti Toxoplasm gondii IgM values in data subsets corresponding to the risk factors that were implemented in the study and odds ratios comparing each level to the first listed within each respective factor